%0 Journal Article
%T Engineering the ¡°Missing Link¡± in Biosynthetic (£¿)-Menthol Production: Bacterial Isopulegone Isomerase
%J -
%D 2018
%R https://doi.org/10.1021/acscatal.7b04115
%X High Resolution Image Download MS PowerPoint Slide The realization of a synthetic biology approach to microbial (1R,2S,5R)-(£¿)-menthol (1) production relies on the identification of a gene encoding an isopulegone isomerase (IPGI), the only enzyme in the Mentha piperita biosynthetic pathway as yet unidentified. We demonstrate that ¦¤5-3-ketosteroid isomerase (KSI) from Pseudomonas putida can act as an IPGI, producing (R)-(+)-pulegone ((R)-2) from (+)-cis-isopulegone (3). Using a robotics-driven semirational design strategy, we identified a key KSI variant encoding four active site mutations, which confer a 4.3-fold increase in activity over the wild-type enzyme. This was assisted by the generation of crystal structures of four KSI variants, combined with molecular modeling of 3 binding to identify key active site residue targets. The KSI variant was demonstrated to function efficiently within cascade biocatalytic reactions with downstream Mentha enzymes pulegone reductase and (£¿)-menthone:(£¿)-menthol reductase to generate 1 from 3. This study introduces the use of a recombinant IPGI, engineered to function efficiently within a biosynthetic pathway for the production of 1 in microorganisms
%U https://pubs.acs.org/doi/10.1021/acscatal.7b04115